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Abstract This study was conducted to screen tomato resources resistant to gray mold for the first time by invitro stem inoculation method. The results showed that Solanum habrochaites T207316 was highly resistant to gray mold， and had the relative stem infection rate and relative stem rot expansion rate of 0 on the 6th day after inoculation. There were also other seven transgenic common tomato materials which showed higher resistance to gray mold， with a relative stem infection rate in the range of 15.00%-38.33% and a relative stem rot expansion rate in the range of 10.22%-23.57%. Among them， T207337 had the best resistance.
Key words Tomato; Gray mold; Screening of resistant resources; Invitro stem inoculation
Tomato gray mold is caused by the infection of Botrytis cinerea Pers. in Deuteromycotina. It is a worldwide important disease， especially serious in tomato cultivated in a protected area. The pathogen can infect the stem， leaf， flower and fruit of tomato， causing serious damage to tomato production[1]. The prevention and control of tomato gray mold has always been an important topic at home and abroad. At present， the prevention and control of gray mold mainly adopts methods such as chemical control and biological control. Although these methods have certain control effects， they also have problems such as unsatisfactory effects， pesticide residues and drug resistance. To completely solve the problem of difficult prevention and control of gray mold， breeding resistant varieties is the most economical and effective means. For a long time， due to the lack of ideal resistant resources， the research on breeding for tomato varieties resistant to gray mold has been developed slowly， and the varieties used in production are all not resistant to gray mold. In this study， tomato resources were screened for gray mold resistance by invitro stem inoculation method， which lays a foundation for next breeding of varieties resistant to gray mold.
Materials and Methods
Materials
The experiment was conducted in 2007 at the Vegetable Research Institute， Liaoning Academy of Agricultural Sciences. The 26 tomato materials and B. cinerea strain tested were provided by the Institute. The 26 tomato materials included 25 identification materials and 1 susceptible control material L402 （code T21）. The 25 identification materials were all from abroad， one of which was S. habrochaites， other 24 were transgenic common tomato， the exogenous gene of which was rice chitinase gene （Table 1）. On August 2， seedlings were raised in plug trays in a greenhouse. When the plants grew with two true leaves， they were planted in the nutrition bowls in the solar greenhouse. The nutrition bowls had a diameter of 9 cm and a height of 9 cm. The used soil was garden soil∶peat∶composted pig manure=3 V∶2 V∶3 V. Each nutrition bowl was planted with one plant. After planting， the temperature was 25 ℃ during the day and 15 ℃ during the night， and watering was performed once every 3 days. Methods
Preparation of B. cinerea liquid
The preparation was performed according to reference[2]. The strain was inoculated on PDA medium， and after the mycelia became dark gray black to form spores， the colony was washed with sterile water to give a spore suspension， which was filtered with gauze， to remove mycelia and obtaining the spore suspension， which was prepared with sterile water to a 1×10 6 spores/ml suspension for artificial inoculation.
Artificial stem inoculation
The inoculation was performed according the method of tenHank et al.[3]. The leaves and growth points of tomato plants at sixleaf stage were cut off. The stems were cut from 5 cm from the base of the stems into 4 cm sections. Each seedling was cut into 3 sections， which were then vertically inserted into incubators filled with sterilized peat and vermiculite （1 V∶1 V）. Before inoculation， the cut stem sections were evenly sprayed with sterile water， and each section was inoculated with 5 μl of spore suspension. After inoculation， the incubators were sealed and stored in a dark condition at a temperature of 20 ℃ and a relative humidity of 100 %. Each material was set with three replications， each of which included 5 sections.
Investigation of incidence
At 4， 5， and 6 d after artificial stem inoculation， the stem infection rate and stem rot expansion rate of the 26 materials were investigated and calculated according to the method of tenHave et al.[3]. The resistance in materials was comprehensively evaluated according to the two indicators.
Stem infection rate=Number of infected sections with disease spots/Total number of inoculated sections×100%
Relative stem infection rate=Stem infection rate of material/Stem infection rate of susceptible control×100%
Stem rot expansion rate （mm/d） = （Length of disease spot on the 6th day after inoculation-Length of disease spot on the 4th day）/2
Relative stem rot expansion rate = Stem rot expansion rate of material/Stem rot expansion rate of susceptible control×100%
Data processing and statistical analysis were performed using Microsoft Excel 2003 and DPS 3.01 software， and multiple comparison analysis was performed by LSD method.
Results and Analysis
Comparison of relative stem infection rate among different materials
It could be seen from Table 1 that among the 26 materials inoculated， the relative stem infection rate of S. habrochaites T207316 was the lowest， and was 0， on the 6th day after inoculation， so it had high resistance to gray mold. T207337 and T207322 had the second highest stem infection rates， which were 15.00% and 20.00%， respectively， on the 6th day after inoculation. T207334， T207315， T207335， T207324， T207 314 and T2 07330 also showed better resistance， and had the relative stem infection rates on the 6th day after inoculation in the range of 23.33%-38.33%， which was significantly lower than that of the susceptible control. Comparison of relative stem rot expansion rate among different materials
It could be seen from Table 1 that among the 26 materials inoculated， the relative stem rot expansion rate of S. habrochaites T207316 was the lowest， and was 0 on the 6th d after inoculation. T207337 and T207315 had the relative stem rot expansion rates of 10.22% and 11.40% on the 6th day after inoculation， respectively. T207322， T207330， T207324， T207334 and T207335 also showed better resistance， and had the relative stem infection rates on the 6th day after inoculation in the range of 13.98%-23.57%， which was significantly lower than that of the susceptible control.
Conclusions and Discussion
At home and abroad， the screening of materials resistant to tomato gray mold has been studied for a long time， but the progress is relatively slow， and few materials have been selected. Farle et al.[4] found that tomato V543 showed certain resistance to gray mold. Egashira et al.[5] found that wild resources such as S. peruvianum LA2745， S. habrochaites LA2314 and S. pimpinellifolium LA1246 showed resistance to gray mold. Nicot et al.[6] also proved that some wild materials， especially S. habrochaites， showed partial resistance to gray mold， and they deem that such condition demonstrates a good prospect for breeding of tomato resistant to gray mold. Guimaraes et al.[7] found that S. lycopersicoides LA2951 also showed high resistance to tomato gray mold， and the hybrid generation was proved to be at least partially dominant. Li[2] identified the disease resistance in 21 wild resource materials of 8 tomato species， and screened 7 resistant tomato materials， i.e.， S. habrochaites PI134417， PI126445， PI247087， LA1392， S. lycopersicoides LA1341， LA2951 and LA2408. It is preliminarily believed that among the wild resource materials， S. habrochaites and S. lycopersicoides can be used as resistance materials against gray mold.
This study screened tomato resources resistant to gray mold for the first time by invitro stem inoculation method. The results showed that S. habrochaites T207316 showed higher resistance to gray mold， and had the relative stem infection rate and relative stem rot expansion rate of 0 on the 6th day after inoculation. This materials was introduced from Germany under the initial code Lyc4. Other seven transgenic common tomato materials also showed higher resistance to gray mold， with a relative stem infection rate in the range of 15.00%-38.33% and a relative stem rot expansion rate in the range of 10.22%-23.57%. Among them， T207337 had the best resistance， and other 6 materials were T207315， T207322， T207334， T207324， T207335 and T207330. In this study， the invitro stem inoculation method was used to screen and identify tomato resources resistant to tomato gray mold. It has the characteristics of simple operation and rapid onset： in smallsized incubators， dozens of samples can be identified， the environmental conditions are also relatively easy to control， and the incidence results can be investigated 6 d after inoculation， indicating significantlyimproved inoculation efficiency. However， invitro stem inoculation requires stricter seedling age. According to tenHave et al.[3]， for 5 to 7 weeks old seedlings， different stem sections had different susceptibility to disease; and for oneweekold plants， top stem section was more susceptible than other stem sections. Therefore， invitro stem inoculation is best to use 5 to 7 weeks old seedlings， in order to obtain more objective and accurate results. In production， tomato gray mold can infect the leaf， stem， flower and fruit of tomato. From the current research progress， the correlation between the gray mold resistance in different tomato parts is still unclear， and therefore， only inoculation of different parts of the plant can more objectively evaluate the resistance level of the tested material.
References
[1] LI BJ， ZHU GR， ZHAO KH， et al. Infected parts of tomato gray mold on fruits and new prevention and treatment techniques[J]. Acta Phytopathologica Sinica， 1999， 29 （1）：63-67. （in Chinese）
[2] LI JM. Mapping of genes resistant to late blight （Phytophthora Infestans） and gray mold （Botrytis cinerea） and multiple gene selection assisted by molecular markers for breeding in tomato （Lycopersicon esculentum）[D]. Beijing： Graduate School of Chinese Academy of Agricultural Sciences， 2005. （in Chinese）
[3] TEN HAVEA， VAN BERLOOR， LINDHOUTP， et al. Partial stem and leaf resistance against the fungal pathogen Botrytis cinerea in wild relatives of tomato[J]. European Journal of Plant Pathology， 2007， 117： 153-166.
[4] FARLEYJD， GEORGEWL， KERREA. Resistance to Botrytis cinerea[J]. Tomato Genetics Cooperative Report， 1976， 26：7.
[5] EGASHIRAH， KUWASHIMAA， ISHIGUROH， et al. Screening of wild accessions resistance to gray mold （Botrytis cinerea Pers.） in Lycopersicon[J]. Acta Physiologiae Plantarum， 2000， 22： 324-326.
[6] NICOTPC， MORETTIA， ROMITIC， et al. Differences in susceptibility of pruning wounds and leaves to infection by Botrytis cinerea among wild tomato accessions[J]. Tomato Genetics Cooperative Report， 2002， 52： 24 -26.
[7] GUIMAR？ ES R， CHETELATRT， STOTZHU. Resistance to Botrytis cinerea in Solanum lycopersicoides is dominant in hybrids with tomato， and involves induced hyphal death[J]. European Journal of Plant Pathology， 2004， 110（1）： 13-23.
Key words Tomato; Gray mold; Screening of resistant resources; Invitro stem inoculation
Tomato gray mold is caused by the infection of Botrytis cinerea Pers. in Deuteromycotina. It is a worldwide important disease， especially serious in tomato cultivated in a protected area. The pathogen can infect the stem， leaf， flower and fruit of tomato， causing serious damage to tomato production[1]. The prevention and control of tomato gray mold has always been an important topic at home and abroad. At present， the prevention and control of gray mold mainly adopts methods such as chemical control and biological control. Although these methods have certain control effects， they also have problems such as unsatisfactory effects， pesticide residues and drug resistance. To completely solve the problem of difficult prevention and control of gray mold， breeding resistant varieties is the most economical and effective means. For a long time， due to the lack of ideal resistant resources， the research on breeding for tomato varieties resistant to gray mold has been developed slowly， and the varieties used in production are all not resistant to gray mold. In this study， tomato resources were screened for gray mold resistance by invitro stem inoculation method， which lays a foundation for next breeding of varieties resistant to gray mold.
Materials and Methods
Materials
The experiment was conducted in 2007 at the Vegetable Research Institute， Liaoning Academy of Agricultural Sciences. The 26 tomato materials and B. cinerea strain tested were provided by the Institute. The 26 tomato materials included 25 identification materials and 1 susceptible control material L402 （code T21）. The 25 identification materials were all from abroad， one of which was S. habrochaites， other 24 were transgenic common tomato， the exogenous gene of which was rice chitinase gene （Table 1）. On August 2， seedlings were raised in plug trays in a greenhouse. When the plants grew with two true leaves， they were planted in the nutrition bowls in the solar greenhouse. The nutrition bowls had a diameter of 9 cm and a height of 9 cm. The used soil was garden soil∶peat∶composted pig manure=3 V∶2 V∶3 V. Each nutrition bowl was planted with one plant. After planting， the temperature was 25 ℃ during the day and 15 ℃ during the night， and watering was performed once every 3 days. Methods
Preparation of B. cinerea liquid
The preparation was performed according to reference[2]. The strain was inoculated on PDA medium， and after the mycelia became dark gray black to form spores， the colony was washed with sterile water to give a spore suspension， which was filtered with gauze， to remove mycelia and obtaining the spore suspension， which was prepared with sterile water to a 1×10 6 spores/ml suspension for artificial inoculation.
Artificial stem inoculation
The inoculation was performed according the method of tenHank et al.[3]. The leaves and growth points of tomato plants at sixleaf stage were cut off. The stems were cut from 5 cm from the base of the stems into 4 cm sections. Each seedling was cut into 3 sections， which were then vertically inserted into incubators filled with sterilized peat and vermiculite （1 V∶1 V）. Before inoculation， the cut stem sections were evenly sprayed with sterile water， and each section was inoculated with 5 μl of spore suspension. After inoculation， the incubators were sealed and stored in a dark condition at a temperature of 20 ℃ and a relative humidity of 100 %. Each material was set with three replications， each of which included 5 sections.
Investigation of incidence
At 4， 5， and 6 d after artificial stem inoculation， the stem infection rate and stem rot expansion rate of the 26 materials were investigated and calculated according to the method of tenHave et al.[3]. The resistance in materials was comprehensively evaluated according to the two indicators.
Stem infection rate=Number of infected sections with disease spots/Total number of inoculated sections×100%
Relative stem infection rate=Stem infection rate of material/Stem infection rate of susceptible control×100%
Stem rot expansion rate （mm/d） = （Length of disease spot on the 6th day after inoculation-Length of disease spot on the 4th day）/2
Relative stem rot expansion rate = Stem rot expansion rate of material/Stem rot expansion rate of susceptible control×100%
Data processing and statistical analysis were performed using Microsoft Excel 2003 and DPS 3.01 software， and multiple comparison analysis was performed by LSD method.
Results and Analysis
Comparison of relative stem infection rate among different materials
It could be seen from Table 1 that among the 26 materials inoculated， the relative stem infection rate of S. habrochaites T207316 was the lowest， and was 0， on the 6th day after inoculation， so it had high resistance to gray mold. T207337 and T207322 had the second highest stem infection rates， which were 15.00% and 20.00%， respectively， on the 6th day after inoculation. T207334， T207315， T207335， T207324， T207 314 and T2 07330 also showed better resistance， and had the relative stem infection rates on the 6th day after inoculation in the range of 23.33%-38.33%， which was significantly lower than that of the susceptible control. Comparison of relative stem rot expansion rate among different materials
It could be seen from Table 1 that among the 26 materials inoculated， the relative stem rot expansion rate of S. habrochaites T207316 was the lowest， and was 0 on the 6th d after inoculation. T207337 and T207315 had the relative stem rot expansion rates of 10.22% and 11.40% on the 6th day after inoculation， respectively. T207322， T207330， T207324， T207334 and T207335 also showed better resistance， and had the relative stem infection rates on the 6th day after inoculation in the range of 13.98%-23.57%， which was significantly lower than that of the susceptible control.
Conclusions and Discussion
At home and abroad， the screening of materials resistant to tomato gray mold has been studied for a long time， but the progress is relatively slow， and few materials have been selected. Farle et al.[4] found that tomato V543 showed certain resistance to gray mold. Egashira et al.[5] found that wild resources such as S. peruvianum LA2745， S. habrochaites LA2314 and S. pimpinellifolium LA1246 showed resistance to gray mold. Nicot et al.[6] also proved that some wild materials， especially S. habrochaites， showed partial resistance to gray mold， and they deem that such condition demonstrates a good prospect for breeding of tomato resistant to gray mold. Guimaraes et al.[7] found that S. lycopersicoides LA2951 also showed high resistance to tomato gray mold， and the hybrid generation was proved to be at least partially dominant. Li[2] identified the disease resistance in 21 wild resource materials of 8 tomato species， and screened 7 resistant tomato materials， i.e.， S. habrochaites PI134417， PI126445， PI247087， LA1392， S. lycopersicoides LA1341， LA2951 and LA2408. It is preliminarily believed that among the wild resource materials， S. habrochaites and S. lycopersicoides can be used as resistance materials against gray mold.
This study screened tomato resources resistant to gray mold for the first time by invitro stem inoculation method. The results showed that S. habrochaites T207316 showed higher resistance to gray mold， and had the relative stem infection rate and relative stem rot expansion rate of 0 on the 6th day after inoculation. This materials was introduced from Germany under the initial code Lyc4. Other seven transgenic common tomato materials also showed higher resistance to gray mold， with a relative stem infection rate in the range of 15.00%-38.33% and a relative stem rot expansion rate in the range of 10.22%-23.57%. Among them， T207337 had the best resistance， and other 6 materials were T207315， T207322， T207334， T207324， T207335 and T207330. In this study， the invitro stem inoculation method was used to screen and identify tomato resources resistant to tomato gray mold. It has the characteristics of simple operation and rapid onset： in smallsized incubators， dozens of samples can be identified， the environmental conditions are also relatively easy to control， and the incidence results can be investigated 6 d after inoculation， indicating significantlyimproved inoculation efficiency. However， invitro stem inoculation requires stricter seedling age. According to tenHave et al.[3]， for 5 to 7 weeks old seedlings， different stem sections had different susceptibility to disease; and for oneweekold plants， top stem section was more susceptible than other stem sections. Therefore， invitro stem inoculation is best to use 5 to 7 weeks old seedlings， in order to obtain more objective and accurate results. In production， tomato gray mold can infect the leaf， stem， flower and fruit of tomato. From the current research progress， the correlation between the gray mold resistance in different tomato parts is still unclear， and therefore， only inoculation of different parts of the plant can more objectively evaluate the resistance level of the tested material.
References
[1] LI BJ， ZHU GR， ZHAO KH， et al. Infected parts of tomato gray mold on fruits and new prevention and treatment techniques[J]. Acta Phytopathologica Sinica， 1999， 29 （1）：63-67. （in Chinese）
[2] LI JM. Mapping of genes resistant to late blight （Phytophthora Infestans） and gray mold （Botrytis cinerea） and multiple gene selection assisted by molecular markers for breeding in tomato （Lycopersicon esculentum）[D]. Beijing： Graduate School of Chinese Academy of Agricultural Sciences， 2005. （in Chinese）
[3] TEN HAVEA， VAN BERLOOR， LINDHOUTP， et al. Partial stem and leaf resistance against the fungal pathogen Botrytis cinerea in wild relatives of tomato[J]. European Journal of Plant Pathology， 2007， 117： 153-166.
[4] FARLEYJD， GEORGEWL， KERREA. Resistance to Botrytis cinerea[J]. Tomato Genetics Cooperative Report， 1976， 26：7.
[5] EGASHIRAH， KUWASHIMAA， ISHIGUROH， et al. Screening of wild accessions resistance to gray mold （Botrytis cinerea Pers.） in Lycopersicon[J]. Acta Physiologiae Plantarum， 2000， 22： 324-326.
[6] NICOTPC， MORETTIA， ROMITIC， et al. Differences in susceptibility of pruning wounds and leaves to infection by Botrytis cinerea among wild tomato accessions[J]. Tomato Genetics Cooperative Report， 2002， 52： 24 -26.
[7] GUIMAR？ ES R， CHETELATRT， STOTZHU. Resistance to Botrytis cinerea in Solanum lycopersicoides is dominant in hybrids with tomato， and involves induced hyphal death[J]. European Journal of Plant Pathology， 2004， 110（1）： 13-23.